Amyotrophic lateral sclerosis (ALS) is an untreatable, paralytic neurodegenerative disorder that is uniformly lethal, usually within 3-5 years. Members of our team have identified mutations in two genes whose mutations cause dominantly inherited familial ALS (fALS): Cu,Zn superoxide dismutase-1 (SOD1), the first identified ALS gene, and FUS/TLS. Respectively, these account for 20% and 5% of fALS cases. Presently, it is not clear how mutant SOD1 or mutant FUS cause fALS; it is likely that the former involves instability of the mutant SOD1 protein and aberrant protein processing, while the latter implicates perturbations of RNA function, as indicated in part by the observation that mutant FUS is often mislocalized to the cytosol. The goal of this proposed project is to discover small molecules that inhibit the cytotoxicity of mutant forms of SOD1 and FUS/TLS. Our hypotheses are that (1) factors that re-direct FUS from the cytoplasm to the nucleus will attenuate FUS- mediated toxicity in ALS patients; and (2) a reduction in the load of toxic mutant-SOD1 proteins will have a therapeutic effect in ALS patients. We have substantial data documenting that our screening and validation assays are functional and thus are confident that our high-throughput screening has the potential to identify small molecules and genetic factors that ameliorate toxicity of mutant forms of both FUS and SOD1. Our study has four aims. Aim 1 is to conduct high throughput screening for compounds that mitigate toxicity of mutant FUS and SOD1 using a yeast model of FUS toxicity (Aim 1A), an HEK293 cell model of FUS mislocalization (Aim 1B) and an HEK model of quantifiable SOD1 expression (Aim 1C). The deliverable from Aim 1 is at least five compounds with efficacy at levels <5 M and toxicity at levels >25 M. Aim 2 is to validate hits from Aim 1, using Drosophila and primary neuronal models of FUS toxicity (Aim 2A) and human cell lines for analysis of SOD1 gene expression. Aim 3 is to optimize the lead compound series and establish structure-activity relationships. The deliverable from Aims 2 and 3 are three compounds from each of the two screening platforms (six total) with efficacy a <1 M and LD50 >50 M that will then be tested in FUS and ALS transgenic mice. Aim 4 is to conduct trials of the six best validated compounds in the transgenic FUS and SOD1G93A mice (three compounds in each). These studies will address two questions. First, do the compound hits achieve anticipated target effects in vivo (for FUS: reduction of FUS-mediated pathology; for SOD1: reduction of SOD1 mRNA and protein levels) (Aim 4A)? And, do the hits ameliorate motor or behavioral abnormalities or prolong survival? (Aim 4B). In our view, this program entails a high degree of innovation both in the assays employed and in the pilot set of compounds discovered so far. We also believe that these investigations hold considerable significance; the need for any meaningful therapeutic intervention in ALS is compelling. Moreover, it is conceivable that the compounds discovered in these studies will prove beneficial in neurodegenerative conditions other than ALS alone. PUBLIC HEALTH RELEVANCE: Amyotrophic lateral sclerosis (ALS) is an untreatable disease that kills motor nerves (which control movement) and causes death in humans within 3-5 years. Our proposed project will focus on two genetic causes of ALS: mutations (or gene defects) in both Cu,Zn superoxide dismutase-1 (SOD1) and Fused in sarcoma/Translocated in liposarcoma (FUS/TLS or FUS). Mutations in these genes account for 25 30% of inherited ALS cases. While we do not fully understand how mutations in SOD1 or FUS cause ALS, we know that reducing the amount of mutant-SOD1 in motor neurons protects these cells from dying, and that the location of FUS in the cell correlates with its toxicity. Normally FUS is concentrated in the nucleus. However mutated FUS is concentrated in the cytoplasm, which surrounds the nucleus. We propose high-through put screens for drug-like molecules that can reduce levels of SOD1 and reduce the toxicity of FUS, possibly by restoring FUS to the nucleus. These drug-like molecules may therefore lead to new treatments for ALS patients.